The present invention relates to the continuous operation of reactors in non-conventional medium, i.e. non-aqueous, in particular for catalytic reactions utilizing essentially a solid phase and a gas phase.
Solid/gas catalytic reactions in which the solid phase of the reactor is constituted by an enzyme and the substrates or products of the reaction are in the form of a gas have been described by Pulvin S., Legoy M. D., Lortie R., Pensa M. and Thomas D. (1986) Enzyme technology and gas phase catalysis: alcohol dehydrogenase example. Biotechnol. Lett., 8, 11, pp 783-784. Catalytic systems which make use of whole cells as constituent elements of the solid phase of the reactor are also known.
Solid/gas catalysis offers in fact certain advantages compared to the conventional systems of the solid/liquid type:
it makes it possible to dispense with the use of solvents and to operate solely with the substrates and the products of the reaction in the immediate environment of the enzyme;
since the solid phase is composed of the biocatalytic element itself, the binding and immobilization steps prove not to be necessary.
the productivity is improved since the mass transfers are large owing to the high diffusivity and the low viscosity of the gas phases.
the gas phase is composed of pure substrates and products and of a vector gas, no solvent is used which facilitates the treatment downstream of the reaction medium.
Solid/gas catalysis requires a higher working temperature than conventional systems. As a result the risks of microbial contamination of the elements of the reactors are less.
The principle of it is the following:
The conversion of a gas substrate optionally transported by a vector gas undergoes conversion at the interface of a solid biocatalyst (enzyme or whole cells), the reaction products being recovered in the form of a gas.
The principles of solid/gas catalysis, the parameters of the reactions concerned are described in Biotechnology and Bioengineering, vol. 45, pages 387-397 (1995).
At present, it has been possible to obtain only a few chemical compounds such as epoxides, aldehydes and esters by solid/gas catalysis systems (ref.). But the principal limit of the existing system is the maintenance of an active biocatalyst and, consequently, its compatibility with industrial use.
A promising application for the reactors of this type relates to the treatment of polluted gas effluents since the range of molecules which need to be eliminated from industrial wastes before their release into the atmosphere is increasing incessantly. They include aldehydes, alcohols, ketones, carboxylic acids, cresols, phenols, sulfur-containing derivatives, cyclic amines, alkanes or esters. An improvement in the depollution procedures of soils may also be achieved by such systems.
However, the solid/gas catalysis systems are related in the first instance by the multiplicity of the constitutive elements which leads to a lack of control of the different parameters, in particular those depending on the complex role of water. In fact, the state of hydration of the enzyme preparation exerts an antagonist effect on the catalytic activity and on the stability of the catalyst with time.
The solid/gas phase reactors developed up to now (Lamare et al., Trends iin Biotechnology (1993) 10 (117): 413-418) are designed to operate at atmospheric pressure, although they are capable of working at temperatures ranging up to 220xc2x0 C. with a controlled thermodynamic activity for each constituent. The definition and importance of the term activity in carrying out the solid/gas enzymatic reaction is described below.
They are inoperative for all the applications in which the components are poorly volatile, i.e. for all the components whose boiling point is situated in the range of 150-250xc2x0 C. Now to-day many reactions capable of being developed because they represent an industrial interest involve compounds whose saturation pressures are low and even close to 0 at the working temperatures used located between 50 and 150xc2x0 C., temperatures compatible with maintenance of the biocatalyst in an active form.
The principal obstacle of the existing systems lies in effecting the transfer to the gas phase of all the components of the system, the reaction substrates and products obtained. No bioreactor permits this conversion. Furthermore, the expense caused by the massive use of a neutral vector gas for a large scale supply to a solid/gas reactor makes the use of this reaction procedure prohibitive for industrial purposes.
Many examples attest to the important role of water in enzymatic catalysis in non-conventional medium. A simple manner of defining the thermodynamic activity of water consists of using the water vapour pressure of the gas phase in equilibrium with the system considered. One may then write:
aw=Pp/Ppref
where Pp is the partial pressure of the water above the system and Ppref the so-called reference partial pressure measured at the same temperature above pure water. The aw of a system is hence a function of physical parameters characterizing a system such as the absolute pressure and the temperature: it is the equilibrium parameter permitting the state of the water to be defined; it allows the influence of water to be unequivocally quantified in a system where the polarity, the dielectric constant of the present chemical entities, the number of phases, the temperature have a considerable influence on the distribution of water in the different phases of the system.
Halling (Halling P. (1984) in Effect of water on equilibria catalysed by hydrolytic enzymes in biphasic reaction systems, Enzyme Microb. Technol., 6, pp 513-515) has illustrated the equilibrium which may exist between the different states of water and the different phases of a complex medium (state of hydration of the biocatalyst and of the other components, quantity of water dissolved in the solvent, partial pressure of water vapour above the system), this equilibrium being a function of the activity of the water.
The value of the thermodynamic activity of the water of a system depends on the physical parameters characterizing this system, such as the absolute pressure and the temperature. The value of the thermodynamic activity of the water is therefore regulated in order to establish the equilibrium operating conditions between the different phases of the reactor; it is thus a decisive parameter for optimizing the reactor and its operating conditions.
The present invention proposes a solution to the problems evoked above by implementing reactions catalysed in the solid/gas phase at reduced pressure in order to optimize the productivity and reduce the expense by minimizing, and even eliminating, the use of a neutral gas vector, by referring to the thermodynamic activities of the water and of the compounds used.
More precisely, the object of the invention is a continuous reaction process by essentially solid/gas catalysis in non-conventional medium, implementing different gas substrates in order to obtain defined products. This process consists of controlling the temperature, which determines the reference saturation pressure of each pure compound, the total pressure of the system and the molar fluxes of the compounds in order to regulate the molar composition of the gas mixture as a function of the values of thermodynamic activity determined for the compounds.
The invention also relates to a reactor featuring suitable means for implementing this process. Such a reactor comprises pumps for controlling the flux of each of the liquid substrates, mass flow meters for the addition of a vector gas and probes for controlling the temperature of an expansion mixer of the substrates in the gas phase, of a reaction chamber comprising a bioreactor containing a biological catalyst and in which the substrates are introduced via a heat exchanger, of the bioreactor and of an analytical sampler at the reaction chamber outlet. A vacuum pump coupled to a vacuum regulation valve is also mounted at the reaction chamber outlet. The pumps, the probes and the valve are connected to a command control coupled to a management processor. As a function of the data received and management algorithms that it applies, the processor transmits in a time-dependent manner command signals to the different organs (pumps, probes and valve) in order to regulate the temperature, the total pressure and the molar fluxes as a function of the values of thermodynamic activity determined.
By biological catalyst is meant any catalyst constituted or derived from a living organism, animal, plant, bacterial, viral or fungal; it may be a whole cell, a cellular organelle, a macro-molecular complex or a molecule, in particular proteins, nucleic acids or mixtures of these latter and exhibiting a catalytic activity.
The process used in the invention makes it possible to increase the productivity of the system compared to a system operating at atmospheric pressure, to minimize or abolish the quantity of vector gas used, to enhance the abundance of the substrates in the gas phase without having to increase the temperature excessively, while diminishing the spatial requirements for a reactor at atmospheric pressure. These advantages may coexist, their respective effects being then modulated.
The possibility to dispense with the vector gas is realized by replacement of this gas by the lowest boiling compound of the compounds introduced in the vapour phase; the thermodynamic activity of this compound may be reduced as a function of the absolute pressure Pa of the system; it then fulfills the function of a vector gas in the process of the invention.
Another advantage of the invention lies in the improvement of the stability of the biological catalyst as a result of the fact that the hydration, on which its thermostability depends, is controlled.
In a particular embodiment and in order to avoid constant modification of the composition of the gas phase during the course of time, the gas to be converted during the reaction results from a mixture of several compounds in liquid form, followed by flash vaporization achieved at high temperature (for example 450xc2x0 C.) with the optional addition of a neutral vector gas after vaporization, if for example it is not desired to reduce the thermodynamic activity of the water.
The reactor according to the invention makes it possible to control the microenvironment of the biocatalyst with precision. It is then possible to cause an enzyme to function and to observe its kinetic behaviour and its solvation/hydration and to validate the modelling of certain interactions, for example protein/ligands interactions.
This reactor opens the door to novel industrial technology where only the effective availabilities of the substrates and water for the enzyme, defined by their thermodynamic activity, are taken into account, making it possible to quantify their effect on catalysis at the molecular level.
The reactor according to the invention as well as the process implemented present many industrial advantages detailed below and which make it possible to:
determine the cost price of the compounds obtained,
enlarge the range of the utilizable substrates and products as well as the range of catalyzable reactions,
use substrates with high boiling points,
control the thermodynamic activities by permitting modifications of free energy of reaction which allows the use of the same catalyst for different reactions including, for example, hydrolysis, transesterification and syntheses in the case of the lipases (example 1 below).
a) Diminution of the Cost Price
The first advantage is apparent in terms of the cost price of the compounds obtained as a result of their implementation for the following reasons: the diminution or abolition of a vector gas simplifies the design of the reactor, reduces the fixed costs of production.
b) Increase of Productivity
The substrate itself can be its own vector which thus enables its relative concentration to be increased and leads to a considerable increase in the productivity of the reaction, i.e. increases the quantity of the products obtained; in fact, the total pressure is reduced to a minimum in order to increase accordingly the n/ntot ratio of each product X, since the partial pressure of each compound is fixed by the value of the thermodynamic activity of said compound. For example, for a conversion at 80xc2x0 C. of a compound with a reference partial pressure of 0.5 atm at this temperature and with a thermodynamic activity of 0.1, the partial pressure of X in the gas to be converted is then equal to 0.05.
For a system operating at atmospheric pressure, the ratio n/ntot is then equal to 0.05. Thus, X represents only 5% of the molar composition of the gas phase.
In a system operating at a reduced absolute pressure of 0.5 atm, the ratio n/ntot necessary to obtain a thermodynamic activity of 0.1 is then equal to 0.1.X thus represents 10% of the molar composition of the gas phase to be converted.
For an operation at constant molar flux in the two systems, the productivity of the system at reduced pressure is multiplied by a factor of two.
The productivity of a system operating at reduced pressure thus leads to a gain in productivity equal to 1/Pabs of the system when these two reactors are compared at constant molar flux.
The reduction of the total pressure of the system also generates a reduction of the amount of vector gas for a given productivity.
In the preceding comparative study, the reduction by half of the total pressure of the system makes it possible to multiply the productivity by two. The choice to obtain an equal productivity makes it possible to feed the reactor with twofold less of the gas phase per unit time. In an embodiment such as the latter, the costs generated by the use of a vector gas such as nitrogen are reduced by a half compared with a system operating at atmospheric pressure.
c) High Boiling Substrates
The reduced pressure reactor of the present invention also allows high boiling substrates to be used. This embodiment xe2x80x9crestrainsxe2x80x9d the thermodynamic activity of water, eliminates completely the necessity for a vector gas and leads to a large increase in the productivity of the reactor.
For example, with the use of a catalyst which necessitates an activity of water of about 0.1, the total pressure of the system is advantageously fixed at 0.1 atm, which corresponds to the partial pressure of water necessary for obtaining an activity equal to 0.1 for a catalysis conducted at 100xc2x0 C. At this temperature the vector gas is constituted solely of water in the form of vapour, within which the substrates are incorporated at up to several matm partial pressure.
In a system defined according to the present invention, the thermodynamic activity of water does not exceed the threshold value of 0.1 even in the case where water is produced by the reaction. The reactor of the present invention avoids any denaturation of the catalyst by an uncontrolled increase of the thermodynamic activity in the system. The productivity of the reactor of the invention for the conversion of the substrates is then multiplied by a factor of 10 compared to a system operating at atmospheric pressure.
d) Displacement of the Reaction Equilibria
The restraint of the thermodynamic activities of certain compounds thus favours the displacements of the reaction equilibrium, while increasing the productivity and diminishing the consumption of vector gas.
The advantages of the solid/gas catalytic reactors operating at reduced pressure make it possible to envisage their use in many fields of economic activity. As examples, mention may be made of:
1) the use of the reactor for the production of organic molecules such as alcohols, carboxylic acids, thiols, thioesters, esters, aldehydes, ketones, alkene oxides, starting from substrates which may be carboxylic acids, primary and secondary alcohols and ketones in particular.
Another aspect of the invention is the use of the solid/gas catalytic reactor for the production of organic molecules cited above. When esters or aldehydes are concerned, obtained by enzymatic conversion of carboxylic acids and alcohols, the products thus obtained can be used as flavours and/or perfumes in the cosmetic or agri-foodstuffs industry, for example. Another advantage of the products thus obtained is that, contrary to those obtained by chemical conversion, they may claim to be natural in conformity with the European directive of Jun. 22, 1988.
Another aspect of the invention is the use of the solid/gas catalytic reactor for the treatment of gas effluents, emanating from industrial processes generating polluted gas phases; in addition to conventional compounds such as SO2, H2S, oxides of nitrogen, mention may be made of aldehydes, alcohols, ketones, carboxylic acids, cresols, phenols, sulfur-containing compounds, cyclic amines, alkanes or esters (Paul Ceccaldi, 1993, Biofutur, No. 126, p. 20).
Another aspect of the invention is the use of solid/gas catalytic reactors for analytical purposes such as the design of enzymatic precolumns for derivatization or acylation for gas chromatography (GC), the development of a gas phase affinity chromatography or the invention of biosensors specific for the detection of volatile molecules (invention of artificial xe2x80x9cnosesxe2x80x9d) are so many directly applicable uses.
Another aspect of the invention is the use of enzymatic reactors in which entire bacterial, animal, plant or fungal cells are used to perform bioconversions. The advantage of the reactor, in this type of use, is that the metabolic activities of the cells in question may be maintained for a sufficiently long period as a result of the control of the thermodynamic activity of water, thus making it possible to perform complex catalytic reactions, in several steps within the same reactor.
Moreover, from the point of view of the use of whole cells in bioconversions, the preparation of the biocatalyst may be achieved in situ and the metabolic activities or their regeneration may be maintained by the control of the thermodynamic activity of water by the process and the reactor according to the invention.